Characterization and epidemiologic analysis of mycoplasmal pneumonia of sheep in Qinghai Province

Mycoplasmal pneumonia in sheep and goats usually result covert but huge economic losses in the sheep and goat industry. The disease is prevalent in various countries in Africa and Asia. Clinical manifestations in affected animals include anorexia, fever, and respiratory symptoms such as dyspnea, polypnea, cough, and nasal discharge. Due to similarities with other respiratory infections, accurate diagnosis can be challenging, and isolating the causative organism is often problematic. However, the utilization of molecular techniques, such as PCR, allows for rapid and specific identification of pathogens. Thus, a goat infection model with Mycoplasma was established and the pathogen was tested using PCR. The results indicated that this approach could be effectively utilized for the rapid detection of mycoplasma in clinical settings. Additionally, the prevalence of contagious pleuropneumonia of sheep in Qinghai Province was further investigated through PCR analysis. A total of 340 nasal swabs were collected from 17 sheep farms in Qinghai province. Among these samples, 84 tested positive for Mycoplasma mycoides subsp. capri (Mmc) and 148 tested positive for Mycoplasma ovipneumoniae (Movi), resulting in positive rates of 24.71% and 43.53% respectively. Furthermore, our investigation revealed positive PCR results for nasal swabs, trachea, and lung samples obtained from sheep exhibiting symptoms suggestive of mycoplasma infection. Moreover, three distinct strains were isolated from these positive samples. Additionally, the inflammatory cytokines of peripheral blood mononuclear cells (PBMCs) were assessed using RT-PCR. The findings demonstrated a high susceptibility of sheep to Movi in Qinghai province, with infected sheep displaying an inflammatory response. Consequently, the outcomes of this study will furnish valuable epidemiological insights for the effective prevention and control of this disease within Qinghai Province.


Introduction
Contagious pleuropneumonia, a widespread disease affecting sheep and goats globally, is caused by pathogens such as Mycoplasma capricolum subsp.capripneumoniae (Mccp), Mmc, and Movi.[1][2][3].Among these, Mccp mainly infect ungulates including sheep, goats, and wild animals [4][5][6], and it is associated with 60% mortality and 90% morbidity in goats [7].The incubation period of the disease is approximately 45 days.In goats, symptoms include anorexia, fever, and respiratory manifestations such as dyspnea, polypnea, coughing, and nasal discharge [8].After sheep become infected with Movi, the resulting clinical symptoms of the disease bear resemblance to those of Mccp infection, characterized by prototypical chronic interstitial pneumonia with significant morbidity and mortality [9].Movi was similar to Mccp, coinfecting with common respiratory pathogens [10].The disease severely impacts both the growth and productivity of lambs [11].Previous reports have identified respiratory disease as the fifth most significant challenge in domestic sheep production within the United States [12].Contagious pleuropneumonia caused by Mccp, Mmc or Movi can lead high mortality rates in goats and sheep, resulting in serious consequences [13].Similarly, they have become increasingly widespread in China.Provinces or districts including Shaanxi, Yunnan, Sichuan, Guizhou, Chongqing, Gansu, Qinghai, Xinjiang and Ningxia have reported its occurrence.The disease has caused significant economic losses in the sheep-farming industry [8,[14][15][16][17][18].
Qinghai Province, being a significant sheep farming region in China, plays a crucial role in the local economy.In recent times, the prevalence of contagious pleuropneumonia of sheep has been observed in various areas of the province, such as Yushu Prefecture, Haiyan County, Guinan County, Pingan County, Huangyuan County, and other counties [19].However, the reports of this disease in Qinghai Province are sporadic.Hence, it is imperative to gain a comprehensive understanding of the current epidemiological situation of contagious pleuropneumonia in Qinghai.Chu et al. [15] previously reported they used a positive indirect hemagglutination test kit to detect antibodies against contagious pleuropneumonia (including Movi, Mmc and Mccp) in Qinghai Province for epidemiological investigation, but antigen testing is slightly less sensitive and precise than PCR testing [20].
The primary aim of this study is to conduct a comprehensive examination of the prevalence of contagious pleuropneumonia in order to facilitate the timely implementation of targeted control and prevention strategies within Qinghai Province.Thus, we performed PCR analysis (including Movi, Mmc and Mccp) of nasal swabs of sheep with suspected contagious pleuropneumonia in different sheep farms of Qinghai province.The findings not only contribute to our understanding of the prevalence of contagious pleuropneumonia, but also offer valuable insights for the prevention of this disease in Qinghai Province.

Bacterial strain and animals
M599 was isolated from a goat farm, and stored in veterinary immunology laboratory (college of veterinary medicine, Northwest A&F University).Three-month-old female goats were purchased locally and housed at the animal center.All goats exhibited no clinical manifestations of disease and were confirmed to be negative for Mccp infection through the utilization of a Mccp detection ELISA kit (Abbexa Ltd, Cambridge, UK).At the end of the protocol, the ritualistic slaughter of sheep was conducted in accordance with the methodologies documented in prior scholarly works [21][22][23].In brief, the sheep were administered i.v.pentobarbital (30 mg/ kg) for anesthesia.After achieving a state of deep anesthesia, followed by i.v.injection of potassium chloride (2 mmol/kg) for euthanasia.The animal experiments were followed by guidelines form Laboratory Animals of the Ministry of Health of China.The study was approved by the Research Ethics Committee of Northwest A&F University.All sample collection was undertaken in accordance with the protocols of the Research Ethics Committee of Northwest A&F University.

Artificial infection of mycoplasma pneumoniae in goats
In order to rapidly detect Mycoplasma in clinical practice, we established a goat infection model with mycoplasma.Culturing methods for lab-isolated strain M599 was the same as reported in our previous study [8].Adjust the concentration of M599 to CCU = 10 9 .The suspension was prepared by diluting the substance in 5 mL of PBS.M599 was infused into the trachea of goats once a day for a week.

Sample collection in Qinghai province
From March to July 2022, 340 nasal swabs were collected from 17 sheep pastures in Qinghai province by randomized stratified sampling.We recorded the age and region of each swab.None of the sheep had been vaccinated against Mycoplasma vaccines.Nasal swabs were composite swabs with the right and left nares, and they were immersed in 3 mL of MEM-KM2 medium.They were kept at 4˚C until DNA extraction.

PCR amplification and the evolutionary tree construction
Swabs were eluted by vigorous agitation in 3 mL of MEM-KM2 medium, centrifuged at 3,500 rpm for 5 min, and the supernatant was collected.200 μL of the supernatant was taken from each sample and lysis-buffer was added to 1 mL respectively.Then they were mixed well, and incubated for 10 min at room temperature.After that, 1 mL of isopropanol solution was added, mixed well, and left for 15 min at -20˚C in a freezer.Next, they were centrifuged at 12,000 rpm for 20 min, and the supernatant was discarded.The PCR reaction system was the same as we reported before [8].The annealing temperatures are 62˚C, 47˚C, and 55˚C, respectively for Mmc, Mccp and Movi.Reaction products were analyzed by 2.0% agarose gel electrophoresis.The PCR products were sequenced in Sangon Biotech (Sangon, Xian, China).The gene sequences were compared with other mycoplasma 16S rRNA gene sequences complete gene sequences by BLAST programs in the GenBank database.The mycoplasma 16S rRNA gene sequences were aligned using MEGA 7.0 software, where 1000 bootstrap replicates were used to determine the nucleotide sequence distance.Table 1 details the primers used in this study.

Isolation of peripheral blood mononuclear cells (PBMCs) of sheep in Qinghai province
Draw 2 mL of blood from the jugular vein in goat.The isolation of PBMCs was performed according to our previous reports [8].Briefly, 2 mL of EDTA treated peripheral blood was centrifuged at 800 g for 5 min, and the supernatant was discarded.The cell pellets were resuspended in 10 mL of ACK Lysis Buffer (Beyotime, Beijing, China) for 10 min at 4˚C.Then, the pellets were centrifuged at 800 g for 5 min, and the supernatant was discarded.Next, they were washed with PBS.After that, the cell pellets were resuspended in 40% Percoll solution (GE Healthcare, Little Chalfont, United Kingdom), and centrifuged at 800 g for 10 min.The pellets were collected as PBMCs.Finally, the PBMCs were resuspended with RNAiso (Takara Bio) at -80˚C until RNA extraction.

The cytokine levels in goat PBMCs were determined using real-time PCR
Total RNA was isolated using RNAiso Plus (Takara), and reverse transcription was used with a PrimeScript RT Reagent Kit (TIANGEN Biotech, Beijing, China).A SYBR Premix Ex TaqTM II kit (TIANGEN) was used for real-time PCR.The reaction system was carried out according to the instructions.Data were collected using a BioRad CFX Real-Time PCR System (Bio-Rad Laboratories, Inc).PCR was performed using the following conditions: 95˚C for 15 s, followed by 40 cycles of 95˚C for 10 s, 60˚C for 30 s. Gene expression levels were analyzed using the 2 −ΔΔCt method.The primers used in this study are listed in S1 Table.

Necropsy and Hematoxylin and Eosin (H & E) staining
At necropsy, lung tissues and trachea were collected.The fresh lung tissues of artificially infected of mycoplasma pneumoniae.Sheep lung specimens without clinical symptoms of mycoplasma were collected in Qinghai province.Next, they were fixed in 4% paraformaldehyde for 2 h, respectively.After that, the tissues were further soaked in fresh 4% formaldehyde for 72 h.Then the tissues were paraffin embedded and cut into 4 μm section slices.Next, the HE staining was performed.The slices were observed under an optical microscope.

Statistical analysis
The data were analyzed using IBM SPSS Statistics.All data were evaluated by one-way ANOVA, and they were presented as the mean ± SEM and representative of at least 2 replicates.If the p-value was less than 0.05, statistical differences were considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).

Artificial infection of Mycoplasma pneumoniae in goats
We performed the PCR on samples from artificially infected goats (Fig 1A).Positive results were found in nasal swab, lung homogenate and tracheal soak solution of infected sheep by PCR.And the PCR products of lung tissue were sequenced, which showed that the goats were infected with the M599 stains (Fig 1B and 1D).At necropsy, pulmonary hemorrhage was observed (Fig 1C).These results suggested that we successfully infected the goats and the PCR method was credible for the detection of mycoplasma sheep in clinical setting.

Detection rate of mycoplasma infection was determined in Qinghai Province
The  For the other information (including breed and gender), there were no significant differences (Fig 6B and 6D).The positive rates of Mycoplasma of nasal swabs were listed by locations, gender, breed, scale, feeding methods and age (S2 Table ).

Clinical manifestations and pathological damage of sheep mycoplasmal pneumonia in Qinghai Province
Typical symptoms include nasal discharge, a high fever (42.6˚C) and poor appetite in clinically infected sheep with Mycoplasma.After dissection, we found that the lung tissues showed pathological changes, including hemorrhage, consolidation, and localized necrosis (Fig 7A).After

Pathogen identification and genetic characterization of M. ovipneumoniae
Three strains were isolated from the positive samples of sheep nasal swabs collected.The strains grew well on the MEM-KM2 medium.After 7-9 days, the white transparent spots of

Discussion
Both sheep numbers and breeds of China are leading in the world, and sheep farming is important for the economies [26].In 2020, the number of goats reached 133.4525 million, and the amount of sheep's depository reached 1730.953 million in China (Data source: www.AskCl.com).Qinghai province contains significant amounts of pasture, and it is one of the five largest animal pastoral areas in China [27].The sheep industry significantly contributes to the economic development of Qinghai province, as evidenced by the increasing volume of sheep's depository and outlet, resulting in substantial economic benefits.However, the prevalence of respiratory disease poses a significant threat to the sheep industry, leading to considerable economic losses.Among these respiratory diseases, Mycoplasma pneumonia stands out as one of the most crucial infectious diseases that jeopardize the production of sheep and goats.This respiratory ailment has been reported in numerous countries, causing substantial economic damage [16,28].One of the diseases that contributes to significant economic losses in endemic regions is contagious caprine pleuropneumonia (CCPP), amounting to approximately US$507 million [17].Additionally, the presence of M. ovipneumoniae infection, even in asymptomatic carriers, has been found to impede the growth rates of lambs [29].This disease is prevalent across various provinces in China and exhibits a tendency to expand.According to relevant reports, which are mainly case descriptions, the incidence and mortality rate of mycoplasma pneumonia in sheep or goats have also shown an upward trend in high-cold areas.This study represents the first investigation into the epidemiology of mycoplasmal pneumonia in sheep within Qinghai province.In the initial phase, we performed artificial infection tests on goats to examine the presence of mycoplasma pneumoniae through PCR analysis of nasal swabs and lung samples.In the current study, our objective was to assess the prevalence of mycoplasma pneumoniae infection in Qinghai Province by employing PCR analysis on nasal swab samples collected from five distinct regions.The findings revealed an average positive rate exceeding 24%, indicating a high prevalence of mycoplasma pneumoniae infection in Qinghai Province.
The average positive rate of M. ovipneumoniae by PCR in this study was 43.53%, which was higher than that found in Xinjiang (M.ovipneumoniae nasal swabs average positive rate was 10.18%), and the overall nasal swab positive rate for M. ovipneumoniae was 40.78% [9,16], whereas lower than the results reported by Xiu-lan, X et al. (the average positive rate was 74%) in Ningxia of China [30].Rong, G et al reported the positive rate of goat serum specimen pneumoniacum was 31.7% in Hainan [31], and Guo, H reported the positive rate of sheep serum was 30.4% in Qinghai Province [15].So, even within the same country, variability may exist in different regions.In Qinghai, the detection rate of mycoplasma pneumonia has shown an increase when compared to data from a decade ago, suggesting a continuous rise in bacterial infection rates.Our survey findings indicate a high detection rate of both Mmc and Movi in lambs, with average positive rates of 29.85% and 45.82% respectively, aligning with previous reports.In comparison, M. ovipneumoniae emerges as the primary infectious agent in sheep [11,16,32].In comparison, M. ovipneumoniae emerges as the primary infectious agent in sheep.Our results show that the Hu sheep has a higher average positive rate (Mmc-32.39%,Movi-41.51%)compared to the other two breeds, which is consistent with a previous report [16].This indicates that sheep breeds have different susceptibilities to M. ovipneumoniae.Alternatively, the average positive rate of Mmc and Movi was 26.01% and 49.4%, respectively in captivity, which is higher than sheep-grazing (the detection rate was 19.09% and 22.74%, respectively).The higher prevalence of mycoplasma pneumonia in captive goats can be attributed to the more crowded conditions they are typically kept in, which serves as a predisposing factor.Additionally, our findings indicate that the rate of detecting the disease in free-ranging sheep (small scale) is greater than in intensively farmed sheep, potentially due to the farmers' professional knowledge and expertise in managing the farming process.Generally, the incidence of mycoplasma pneumonia in sheep is influenced by various factors such as disease outbreaks, breed, location, environment, age, and stress levels.Furthermore, the detection rates of the disease have been observed to correlate with the sample size of studies or the number of affected cases.Based on the findings, it can be inferred that the susceptibility of sheep in Qinghai Province to mycoplasma may be influenced by factors such as age, gender, and rearing condition.

Table 1 . 13 ]
Detailed information for the primers used listed.Mc Auliffe et al. 2003 5-GACTTCATCCTGCACTCTGT-3 https://doi.org/10.1371/journal.pone.0299928.t001 positive detection rate of Mycoplasma (include Mmc and Movi) infection was determined by PCR using samples collected from sheep in Qinghai Province.(As the positive detection rate of Mccp is very low, therefore, the data were not shown).In order to make the experimental results more clear, we plotted the sampling geographical location and labeled the positive detection rate (Fig 2).PCR showed that some samples of nasal swab showed positive bands (Fig 3A, 3B and S1 Fig).The statistical analysis demonstrates, among the 340 tested nasal swabs, 84 Mmc positives and 148 Movi positives were detected.The positive rates were 24.71% and 43.53%, respectively.And the positive rate of Movi was statistically significantly higher than that of Mmc (Fig 4A).For Mmc, the positive rate of sheep aged 0-6 months was

Fig 1 .
Fig 1.The detection assay was established for mycoplasma.(A) Standard strains (F38, Y98, and M599.The lengths of the amplified products of Mmc, Mccp and Movi were 548, 316 and 361 bp, respectively.M: DNA marker, P: positive control (standard strains F38), 1: M599, P1: positive control (standard strain Y98) and N: negative control (double distilled water).(B) PCR-based analysis of DNA samples of infected goats with M599.M: DNA marker, P: positive control (M599), 1: nose swab, 2: lung homogenates, 3: soaking solution of the trachea, N: negative control (double distilled water).(C) Representative images of the pulmonary tissues of the healthy and infected goats with M599.The yellow arrows show hemorrhaging of lung tissue.(D) DNA from infected goat lung tissue was subjected to PCR amplification, and the resulting products were sequenced.https://doi.org/10.1371/journal.pone.0299928.g001

Fig 2 .
Fig 2. Map of the sample collection sites.The figure illustrates the geographical distribution of collected samples in Qinghai Province and the prevalence rate of mycoplasma infection in sheep.This map was drawn on the https://www.ichartcool.com/my.html website.(Disclaimer: The author of this article uploaded the data in the local map and created the final presentation of the map, ensuring that no copyright issues were involved.The basemaps used in the construction of map images were acquired from the Ministry of Natural Resources of the People's Republic of China.Drawing approval number: GS(2019)3333).https://doi.org/10.1371/journal.pone.0299928.g002

Fig 3 .Fig 4 .
Fig 3.The PCR amplification products of DNA extracted from a partial sheep nose swab were subjected to agarose gel electrophoresis.(A) PCR-based analysis of DNA samples of the nose swab were detected in sheep with Mmc.M: DNA marker, P: positive control (standard strains F38), N: negative control (double distilled water), 1-13: Nose swab samples.(B) The nose swab samples were detected in sheep with Movi by PCR.M: DNA marker, P: positive control (standard strains Y98), N: negative control (double distilled water), 1-16: Nose swab samples.https://doi.org/10.1371/journal.pone.0299928.g003

Fig 5 .Fig 6 .
Fig 5.An analysis was conducted on the PCR positive detection rate of Mmc in sheep nasal swabs collected from Qinghai Province.(A) Statistical analysis categorized by age.(B) Statistical analysis categorized by breed.(C) Statistical analysis categorized by scale.(D) Statistical analysis categorized by gender.(E) Statistical analysis categorized by feeding methods.* P < 0.05.https://doi.org/10.1371/journal.pone.0299928.g005

Fig 7 .
Fig 7. Clinical symptoms and pathological lesion of sheep infected mycoplasma in Qinghai province.(A) The clinical manifestation is the nose with nasal fluid and mental depression.The pathological changes are lung tissue hemorrhage, consolidation and localized necrosis.(B) Specificity of the PCR for bacterial cultures, M: DNA marker, P: positive control (standard strains Y98), N: negative control (double distilled water), 1: nose swab, 2: lung homogenates, 3: soaking solution of the trachea.(C) Lung histology sections.The yellow arrows show neutrophil infiltration, and the red arrow shows distension of alveoli, alveolar rupture and fusion forming pulmonary bullae.https://doi.org/10.1371/journal.pone.0299928.g007